**2.1 Proteins responsible for white wine protein instability**

Several studies were performed concerning wine protein instability. Koch and Sajak [18] verified using electrophoresis that the heat-formed deposits enclosed two types of protein fractions with diverse heat sensitivities. Moretti and Berg [19] after fractionation and analysis of wine proteins concluded that, among grape and wine proteins, that the protein fractions with low isoelectric points and low molecular weights were more sensitive to heat treatment and responsible for wine protein instability. The main proteins related to the white wine protein instability have a low molecular weight (12.6–30 kDa) and isoelectric point (4.1–5.8) and contain glycoproteins [20]. Waters et al. [21] did the separation and the fractionation of wine proteins using a combination of salting out with ammonium sulphate and ultrafiltration, showing that the protein fractions with those characteristics (24 and 32 kDa) were more sensitive to high temperatures, contributing more to the white wine protein instability. The lower-molecular-weight protein fractions appear to be the major responsible for white wine haze, where the protein with 24 kDa produced nearly 50% more haze with identical concentration than protein fractions with 32 kDa [13]. Many works showed that pathogenesis-related proteins (PR) are the principal responsible for wine protein instability [13, 22]. Pathogenesis-related proteins are very important for plant protection and are associated with its disease resistance, growth and adaptation to stressful environments [23]. *Vitis vinifera* is the most used for winemaking; however, it is very sensitive to pathogens, particularly fungi and oomycetes, such as *Botrytis cinerea* and *Plasmopara viticola*, respectively [24]. Pathogenesis-related proteins (PR) are produced by the plant in response to infection by pathogens [25], to control the harm made to the grapevine [26]. In *V. vinifera* grape varieties, the thaumatin-like (PR-5 type, 24 kDa protein fraction) [13] and chitinases (PR-3 type, 28 kDa protein fraction) [13] are the two main pathogenesis-related (PR) proteins separated from wine, presented a globular structure and at wine pH a positive charge [27]. Other examples of PR proteins existing in lesser quantity in wine are osmotins, β-1,3-glucanases, invertases, lipid transfer proteins [28]; however, diverse isoforms of thaumatin-like proteins and chitinases have been recognised in grape musts of several *V. vinifera* grape varieties, with a molecular weight between 20 and 30 kDa, and an isoelectric point between 3.0 and 5.0 [7, 29]. These are the principal soluble proteins from *V. vinifera* [22, 30] and are responsible for haze development in bottled white wine during storage and transportation [27, 31]. These proteins are synthesised

#### *White Wine Protein Instability: Origin, Preventive and Removal Strategies DOI: http://dx.doi.org/10.5772/intechopen.101713*

during grape development in function of the grape variety [32], region and year [24, 28] presenting higher levels in the ripening; that means that riper grapes are the more susceptive to protein instability [30]. Chitinases and thaumatin-like proteins present a significant amount of disulphide bonds that contribute to their chemical stable structures and some resistance during the vinification process (some resistance at the low grape juice pH (3.0–3.8)) and resistance to proteolysis [5, 33]. However, the low-molecular-weight proteins (chitinases) are sensitive to temperature changes [22] and wine pH [34]. Thaumatin-like proteins are more thermostable and insensible to wine pH variations, showing no significant structural variations or aggregation in different wine pH [34]. The different sensibility of chitinases and thaumatin-like proteins appears to be associated with the differences in the secondary structure of both proteins, elliptical for chitinases and globular for thaumatin-like proteins [34, 35]. The pathogenesis-related proteins are present in different concentrations in the grape juice of the diverse grape varieties of Sultana, Sauvignon Blanc, Pinot Noir, Muscat of Alexandria and Shiraz with chitinases/thaumatin-like proteins of 118/119, 76/119, 44/23, 21/35 and 9/18, respectively [30]. These researchers likewise verified that grape berry destruction throughout mechanical grape picking, related to long-distance transportation, could encourage the production of pathogenesisrelated proteins by the grape defence mechanism before grape pressing [36]. In fact, chitinases and thaumatin-like proteins and its variances in heat stability give the impression that protein composition may influence haze development in wines [9, 37]. Thaumatin-like protein (24 kDa fraction) is the principal responsible for haze formation relative to chitinase (32 kDa fraction) [21, 38]. However, chitinases are very sensitive to precipitation, where a high correlation was verified between wine chitinases levels and wine haze obtained [37]. The thaumatin-like proteins shows a melting temperature of 62°C, with a determined denaturation half-life of 300 years at 25°C, and chitinases present a denaturation half-life of 6 minutes at 55°C, consequently extrapolating down to a denaturation half-life of 3 days at 35°C or 2 years at 25°C. It was observed that vacuolar invertase (GIN1), from the grapes, and β-(1–3)-glucanases (32 kDa fraction) can also influence haze formation. In fact, the existence of *V. vinifera* thaumatin-like protein bands, β-(1,3)-glucanase and maturation-related protein-like (27.4 kDa) Grip22 precursor have been associated with the natural protein haze of white wines [13, 39, 40]. In fact, there is not a correlation between the total amount of protein and wine protein instability [39].
